The integrin superfamily includes at least 24 family members consisting of heterodimers that utilize 18 alpha and 8 beta chains (Hynes, (2002) Cell 110: 673-87). This family of receptors is expressed on the cell surface and mediates cell-cell and cell-extracellular matrix interactions that regulate cell survival, proliferation, migration, and differentiation as well as tumor invasion and metastasis (ffrench-Constant and Colognato, (2004) Trends Cell Biol. 14: 678-86). Integrins bind to other cellular receptors, growth factors and extracellular matrix proteins, with many family members having overlapping binding specificity for particular proteins. This redundancy may ensure that important functions continue in the absence of a particular integrin (Koivisto et al., (2000) Exp. Cell Res. 255: 10-17). However, temporal and spacial restriction of expression of individual integrins with similar specificity has also been reported and may alter the cellular response to ligand binding (Yokosaki et al., (1996) J. Biol. Chem. 271: 24144-50; Kemperman et al., (1997) Exp. Cell Res. 234: 156-64; Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10).
The integrin family can be divided into several sub-families based on ligand specificity of the heterodimers. One subfamily consists of all of the integrins that recognize and bind the RGD tripeptide. These receptors include the αIIb/β3 and all of the αV heterodimers (Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10). While the αV chain can pair with 5 known beta chains, several of these beta chains can only pair with αV. The β6 chain is selective for heterodimerization to αV and this pair binds extracellular matrix and cytokine proteins with either high or low affinity. αVβ6 binds to the RGD motifs on both TGFβ1LAP and TGFβ3LAP latent complexes and activates them (Munger et al., (1999) Cell 96: 319-328; Annes et al., (2002) FEBS Letters 511: 65-68). However, it does not bind to or activate TGFβ2LAP, which does not have the tri-peptide (Ludbrook et al., (2003) Biochem. J. 369: 311-18). αVβ6-mediated activation of TGFβ requires the latent TGFβ binding protein 1 (LTBP1), which tethers the latent TGFβ complex to the extracellular matrix. Activation is proposed to result from a conformation change induced as the TGFβLAP is held between the cell and the matrix by αVβ6 and LTBP1, respectively (Keski-Oja et al., (2004) Trends Cell Biol. 14: 657-659; Annes et al., (2004) J. Cell Biol. 165: 723-34). The picoMolar binding affinity of αVβ6 for the TGFβLAP complexes is the highest for any of its known ligands. Other ligands for αVβ6 include fibronectin, tenascin, vitronectin and osteopontin (Busk et al., (1992) J. Biol. Chem. 267: 5790-6; Prieto et al., (1993) PNAS 90: 10154-8; Huang et al., (1998) J. Cell. Sci. 111(Pt 15): 2189-95; Yokosaki et al., (2005) Matrix Biol. 24: 418-27). The binding affinity of αVβ6 for these extracellular matrix proteins is low affinity and in the nanoMolar range.
Expression of αVβ6 integrin is restricted to areas of active tissue remodeling in the adult, specifically on the epithelia of healing wounds and at the edge of invading tumors (Breuss et al., (1995) J. Cell Sci. 108: 2241-51). Keratinocytes at the wound edge upregulate the expression of αVβ6 during their migration into the wound, but expression remains high after the edges of the wound epithelium have joined (Breuss et al., (1995) J. Cell Sci. 108: 2241-51; Haapasalmi et al., (1996) J. Invest. Dermatol. 106: 42-48). The wound extracellular matrix contains fibronectin, tenascin and vitronectin, all of which are ligands for αVβ6 (Busk et al., (1992) J. Biol. Chem. 267: 5790-6; Koivisto et al., (1999) Cell Adhes. Commun. 7: 245-57; Hakkinen et al., (2000) J. Histochem. Cytochem. 48: 985-98). In addition, αVβ6 upregulates the expression of the matrix metalloproteinase, MMP-9, that can degrade Type IV collagen and promote cell movement (Niu et al., (1998) Biochem. Biophys. Res. Com. 249: 287-91; Agrez et al., (1999) Int. J. Can. 81: 90-97; Thomas et al., (2001) Int. J. Cancer 92: 641-50; Gu et al., (2002) Br. J. Can. 87: 348-51). Based on its expression pattern in wounds and in vitro studies, αVβ6 may have dual roles to promote keratinocyte migration during wound closure and later to resolve the wound through the activation of TGFβ. The activation of TGFβ by αVβ6 would contribute to wound resolution through the regulation of re-epithelialization, suppression of inflammation and promotion of connective tissue regeneration and scar formation (Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10). In vivo wound studies using beta 6 null mice indicated that wounds healed but there was a markedly increased inflammatory response in the skin. Wound closure and keratinocyte activity was likely unaffected by the loss of αVβ6 because of the expression of other integrin family members (Huang et al., (1996) J. Cell Biol. 133: 921-8). The inflammatory infiltrate in the beta 6 null mouse wounds resembled those from TGFβ1 null mice, suggesting that there was insufficient activity of this cytokine to suppress the immune response in the absence of αVβ6 (Shull et al., (1992) Nature 359: 693-9; Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10).
Analysis of the beta 6 null mice in lung injury and kidney disease models has also identified a role for αVβ6 in fibrosis. Lung fibrosis in the beta 6 null mice was inhibited in a bleomycin injury model (Munger et al., (1999) Cell 96: 319-328). These animals also were protected from an MMP12 dependent emphasema-like phenotype (Morris et al., (2003) Nature 422: 169-73). Both disease phenotypes are dependent on the activation of TGFβ (Munger et al., (1999) Cell 96: 319-328). Inhibition of αVβ6 integrin-mediated TGFβ activation was also hypothesized to promote pulmonary edema in the early phase response to acute lung injury (Pittet et al., (2001) J. Clin. Invest. 107: 1537-44). Beta 6 null mice were also protected from fibrosis in a kidney disease model, where TGFβ activation is essential for the development of tubulointerstitial fibrotic lesions (Ma et al., (2003) Am. J. Pathol. 63: 1261-73).
In addition to its expression in wound healing, the αVβ6 integrin is upregulated at the periphery of many human tumors. αVβ6 expression has been reported in oral (Breuss et al., (1995) J. Cell Sci. 108: 2241-51; Jones et al., (1997) J. Oral Pathol. Med. 26: 63-8; Hamidi et al., (2000) Br. J Cancer 82: 1433-40; Regezi et al., (2002) Oral Oncology 38: 332-6; Impola et al., (2004) J. Pathol. 202: 14-22) and skin squamous cell carcinomas, as well as carcinomas of the lung (Smythe et al., (1995) Can. Met. Rev. 14: 229-39), breast (Arhiro et al., (2000) Breast Can. 7: 19-26), pancreas (Sipos, et al., (2004) Histopathology 45: 226-36), stomach (Kawashima et al., (2003) Pathol. Res. Pract. 199: 57-64), colon (Bates et al., (2005) J. Clin. Invest. 115: 339-47), ovary (Ahmed et al., (2002) Carcinogenesis 23: 237-44; Ahmed et al., (2002) J. Histochem. Cytochem. 50: 1371-79), and salivary gland (Westernoff et al., (2005) Oral Oncology 41: 170-74). In many of these reports the expression of αVβ6 correlated with increasing tumor grade (Ahmed et al., (2002) J. Histochem. Cytochem. 50: 1371-79; Arihiro et al., (2000) Breast Can. 7: 19-26), eventual metastases to lymph nodes (Kawashima et al., (2003) Pathol. Res. Pract. 199: 57-64; Bates et al., (2005) J. Clin. Invest. 115: 339-47), or poor prognosis (Bates et al., (2005) J. Clin. Invest. 115: 339-47). The most well studied tumor type is oral squamous cell carcinoma, where investigators have also examined αVβ6 in pre-cancerous lesions and correlated its expression with progression to malignancy (Hamidi et al., (2000) Br. J Cancer 82: 1433-40). The link between αVβ6 expression and tumor progression has also been investigated in colon carcinoma where the presence of integrin correlated with the epithelial-to-mesenchymal transition (EMT) of colon cells in an in vitro model (Brunton et al., (2001) Neoplasia 3: 215-26; Bates et al., (2005) J. Clin. Invest. 115: 339-47). The EMT is a normal developmental process that enables epithelial cells to leave their home tissue and migrate out to new areas (Thiery and Sleeman, (2006) Nat. Rev. Mol. Cell Biol. 7: 131-42). It is marked by an increase in the expression of proteins that promote the migration and invasion of cells, such as matrix proteases, cytokines like TGFβ and a variety of cellular adhesion molecules, including integrins (Zavadil and Bottinger, (2005) Oncogene 24: 5764-5774). The expression of EMT markers has also been identified in tumors, particularly in aggressively invasive and metastatic carcinomas. The ability of αVβ6 to promote adhesion to interstitial fibronectin, upregulate the expression of MMP-9 and other matrix proteases and to activate TGFβ indicates it may facilitate the EMT of malignant cells and tumor progression (Bates and Mercurio, (2005) Cancer Bio. & Ther. 4: 365-70).
Animal and in vitro models of human cancer have implicated αVβ6 mediated signal transduction in the promotion of cell proliferation and inhibition of apoptosis. The residues within the C-terminus of the beta 6 chain that promote proliferation of αVβ6-transfected SW480 colon tumor cells in a collagen gel matrix in vitro were identified. Compared to the full-length β6 transfected SW480 cells, the β6 deletion mutant had markedly reduced ability to grow sub-cutaneously in Nude mice (Agrez et al., (1994) J. Cell. Biol. 127: 547-56). In an oral cancer cell line that stably expressed αVβ6, binding to fibronectin resulted in the recruitment and activation of the Fyn kinase by the beta 6 subunit. Downstream signal transduction resulted in the production of MMP-3, promoted cell proliferation in vitro, tumor invasion in an orthotopic model, and metastasis in a tail vein injection model (Li et al., (2003) J. Biol. Chem. 278: 41646-53).
Suppression of apoptosis, like cell proliferation, is another way that αVβ6 may promote tumor growth. Normal stratified squamous epithelia express the αVβ5 integrin but down-regulate it and upregulate αVβ6 expression upon transformation to carcinomas. Using carcinoma cell lines that over-expressed αVβ5, αVβ6 expression was shown to prevent suspension-induced cell death (anoikis) in vitro (Janes and Watt, (2004) J. Cell Biol. 166: 419-31). Apoptosis inhibition has also been observed in vitro in ovarian cancer cell lines treated with cisplatin, which may represent a mechanism for drug resistance of these tumors in vivo (Wu et al., (2004) Zhonghua Fu Chan Ke Za Zhi 39: 112-14).
A number of investigators have developed therapeutics to target αVβ6 activity in fibrosis and cancer. A murine antibody with specificity for the αVβ6, αVβ3 and αVβ5 integrins was shown to prevent adhesion of HT29 colon carcinoma cells to vitronectin and fibronectin in vitro (Lehmann et al., (1994) Can. Res. 54: 2102-07). Another murine antibody therapeutic specific for the human αVβ6 protein was demonstrated to inhibit the invasive growth of HSC-3 oral carcinoma cells in a transoral xenograft tumor model in mice (Xue et al., (2001) Biochem. Biophys. Res. Com. 288: 610-18). A series of human αVβ6 specific antibodies were raised using the beta 6 null mouse model as the host. These antibodies were able to block both TGFβLAP and fibronectin binding to integrin in vitro (Weinreb et al., (2004) J. Biol. Chem. 279: 17875-87). They also demonstrated significant tumor growth inhibition in a human pharyngeal cancer xenograft model (Leone et al., (2003) Proc. of the Am. Assoc. Can. Res. 44, Abstract #4069).
In addition to function blocking antibodies the creation of a peptidomimetic inhibitor of the human αVβ6 integrin has been reported. This compound was shown to inhibit UCLAP-3 cell binding to fibronectin with an IC50 in the 200 nM range with additional activity to block αVβ5 and αVβ3 integrin-mediated cell binding to vitronectin in the 3-20 uM range, respectively (Goodman et al., (2002) J. Med. Chem. 45: 1045-51).
Another recently described role for αVβ6 is as a cellular receptor for viral pathogens. It mediates the binding of the viral capsid for foot-and-mouth disease virus and the Coxsackievirus 9 to enable viral entry in vitro (Miller et al., (2001) J. Virol. 75: 4158-64; Williams et al., (2004) J. Virol. 78: 6967-73). Both foot-and-mouth disease virus and Coxsackievirus 9 capsid proteins contain an RGD sequence that is recognized by multiple integrin family members. Viral entry of both pathogens is blocked by antibody to αVβ6 integrin (Williams et al., (2004) J. Virol. 78: 6967-73).